Methods and pharmaceutical compositions for the treatment of non-alcoholic steatohepatitis

ABSTRACT

In one aspect, methods of treating non-alcoholic steatohepatitis (NASH) or preventing or delaying the progression of non-alcoholic fatty liver disease (NAFLD) to NASH are provided. In some embodiments, the method comprises administering a therapeutically effective amount of ubenimex.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/400,963, filed Sep. 28, 2016; to U.S. Provisional PatentApplication No. 62/460,606, filed Feb. 17, 2017; and to U.S. ProvisionalPatent Application No. 62/469,722 filed Mar. 10, 2017, the entirecontent of each of which is incorporated by reference herein for allpurposes.

FIELD OF THE INVENTION

The present disclosure provides methods and compositions for thetreatment of non-alcoholic steatohepatitis (NASH) and prevention ofprogression to fibrosis and cirrhosis of the liver and hepatocellularcarcinoma (HCC) resulting therefrom, and so relates to the fields ofmedicine, medicinal chemistry, pharmacology, chemistry, and biology.

BACKGROUND OF THE INVENTION

Fatty liver, also known as fatty liver disease (FLD) or hepaticsteatosis, is a reversible condition in which large vacuoles oftriglyceride fat accumulate in liver cells via the process of steatosis(i.e., abnormal retention of lipids within a cell). Despite havingmultiple causes, fatty liver is most commonly associated with excessivealcohol consumption and obesity. FLD associated with other diseases thatinfluence fat metabolism is referred to as “non-alcoholic” FLD or“NAFLD.”

Fatty change represents the intracytoplasmatic accumulation oftriglycerides (neutral fats). At the onset of FLD, the hepatocytespresent small fat vacuoles (liposomes) around the nucleus(microvesicular fatty change). In late stages of FLD, the size of thevacuoles increase and vacuoles coalesce to produce irreversible fattycysts or lesions. Liver disease with extensive inflammation and a highdegree of steatosis often progresses to more severe forms of thedisease.

Non-alcoholic steatohepatitis (NASH) is an extreme and progressive formof NAFLD that is not linked to alcohol consumption and is furtheraccompanied by inflammation (hepatitis). NASH is accompanied byballooning degeneration of hepatocytes (also referred to herein as“hepatocyte ballooning”), which refers to the increase in size (i.e.,ballooning) of cells during this process that is considered to be a formof apoptosis. Ballooned cells are typically two to three times the sizeof adjacent hepatocytes and characterized by a wispy cleared cytoplasmon H&E stained sections. Liver cell death and the inflammatory responselead to activation of stellate cells, which play a pivotal role inhepatic fibrosis. Further disease progression leads to cirrhosis andhepatocellular carcinoma (HCC), resulting in liver failure and,ultimately, death.

For patients suffering from early stages of NASH, lifestyleintervention, such as significant weight reduction, may slow or evenreverse the process of steatosis. However, for patients with advancedNASH, there are no currently available therapies. For example, a recentclinical study of cenicriviroc for the treatment of advanced NASH failedto meet its primary endpoint of improving inflammation and liver damageafter a year of treatment. Given the severity of FLD and NASH and unmetclinical need, an effective therapeutic treatment is urgently needed.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides methods, materials, andpharmaceutical compositions, for treating and preventing the progressionof FLD and NASH. It is envisaged that the methods and compositionsdescribed herein can slow or prevent NAFLD patients from progressing toNASH and can be used to treat NASH patients with beneficial effects ofslowing, stopping, or reversing NASH disease progression in thosepatients

In one aspect, the present disclosure relates to pharmaceuticalcompositions and methods for oral delivery of ubenimex((2S)-2-[[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]amino]-4-methylpentanoicacid; also known asN-[(2S,3R)-3-Amino-2-hydorxy-4-phenylbutyryl-L-leucine, or Bestatin™ asmarketed by Nippon Kayaku in Japan) to treat FLD and NASH patients andto prevent and/or slow the progression of the disease to fibrosis,cirrhosis, HCC, and death. In many embodiments, these compositions fororal administration are formulated as immediate release preparations,e.g. tablets, capsules, or pills, and are conveniently packaged, forexample, in the form of the pill bottles or blister packaging, forpatients or their care providers to administer in therapeuticallyeffective amounts.

In some embodiments, ubenimex is administered in the treatment of NASHin accordance with the disclosure by oral administration of dosesranging from as little as 5 mg per day to up to 500 mg per day at dosingfrequencies of once (QD administration), twice (BID administration), orthrice per day (TID administration). In some embodiments, administrationof doses of from about 30 mg to about 150 mg administered from once tothrice daily is efficacious in the treatment of the majority of FLD andNASH patients, although lower doses can be efficacious in some patients.

In some embodiments, a patient receives continuous daily dosing ofubenimex, with a patient taking a therapeutically effective dose atleast once per day for an extended period of time. Measureablesymptomatic improvement or a detectable slowing of disease progressionmay not occur until after several weeks or more of treatment. Clinicaltrials to demonstrate efficacy will likely run for at least 12 and morelikely 48 weeks of treatment. Accordingly, patients may be treated formultiple consecutive days, weeks (e.g., at least 12, 24, 36, or 48weeks), months (e.g., at least 4 or 6 months), or years, including forthe rest of the patient's life.

In some embodiments, a patient is administered one or more additionalmedications in a combination therapy for at least some portion of thetime they receive ubenimex therapy. Such additional medications include,without limitation, a farnesoid X receptor (FXR) agonist (including butnot limiting to obeticholic acid); a peroxisome proliferator-activatorreceptor (PPAR) agonist, e.g., a PPAR-alpha agonist, a PPAR-beta/deltaagonist, or a PPAR-gamma agonist (including but not limited toelafibranor); aramchol; a caspase inhibitor (including but not limitedto emricasan); a galectin 3 inhibitor (including but not limited toGR-MD-02, Galectin Therapeutics); a MAPK5 inhibitor (including but notlimited to GS-4997, Gilead Sciences); an FGF21 agonist (including butnot limited to BMS-986036, Bristol-Myers Squibb); a LTD4 receptorantagonist (including but not limited to tipelukast); a niacin analog(including but not limited to ARI 3037 MO, Arisaph Pharmaceuticals); anASBT inhibitor (including but not limited to volixibat); an apoptosissignal regulating kinase 1 (ASK1) inhibitor; an angiotensin convertingenzyme (ACE) inhibitor; an angiotensin receptor blocker (ARB, includingbut not limited to telmisartan); a chemokine receptor inhibitor (e.g.CCR2 and/or CCR5 inhibitors, including but not limited to cenicriviroc);a thiozolidinedione (including but not limited to rosiglitazone andpioglitazone); a GLP-1 analog (including but not limiting tosemaglutide, liraglutide); and a biguanide (including but not limited tometformin). In some embodiments, such treatments in accordance with thedisclosure can improve insulin sensitivity in adipose tissue byactivating PPAR and generate biochemical and histological improvementsin NASH.

In some embodiments, methods for treating NASH are provided, saidmethods comprising administration of ubenimex to a patient in need oftreatment. In some embodiments, the ubenimex is administered orally. Insome embodiments, the ubenimex is administered at a daily dosage of 1000mg or less. In some embodiments, the daily dosage is from about 5 mg toapproximately 450 mg. In some embodiments, the daily dosage is in therange of 5-450 mg and is administered QD, BID, or TID, wherein the dailydosage is administered using unit dosage forms comprising ubenimex in anamount of 5, 10, 15, 30, 60, 75, 90 or 150 mg. In some embodiments, thedaily dosage is administered BID and each dose is administered at a doseof 250 mg, 240 mg, 225 mg, 210 mg, 200 mg, 180 mg, 150 mg, 125 mg, 120mg, 100 mg, 90 mg, 75 mg, 60 mg, 50 mg, 30 mg, 15 mg, 10 mg, or 5 mg. Insome embodiments, the daily dosage is administered TID and each dose isadministered at a dose of 150 mg, 125 mg, 120 mg, 100 mg, 90 mg, 75 mg,60 mg, 50 mg, 30 mg, 15 mg, 10 mg, or 5 mg. In some embodiments, theubenimex is administered for at least 4 weeks. In some embodiments,treatment reduces fibrosis in the patient. In some embodiments,treatment reduces hepatocyte ballooning in the patient. In someembodiments, treatment reduces inflammation in the patient.

In some embodiments, methods for reducing hepatocyte ballooning in aNAFLD or NASH patient are provided, said method comprising administeringubenimex at a daily dosage of from 5 to 450 mg using consecutive dailydosing for at least 4 weeks. In some embodiments, the ubenimex isadministered for at least 12 weeks.

In some embodiments, methods for decreasing inflammation and/or fibrosisin a NASH patient are provided, said method comprising administeringubenimex at a daily dosage of from 5 to 450 mg using consecutive dailydosing for at least 24 weeks. In some embodiments, the ubenimex isadministered for at least 48 weeks.

In some embodiments, methods for the treatment or prevention ofnon-alcoholic steatohepatitis or its progression are provided, saidmethod comprising administration of ubenimex and optionally a secondarypharmaceutical agent to a patient in need thereof. In some embodiments,the secondary pharmaceutical agent is a farnesoid X receptor (FXR)agonist, a peroxisome proliferator-activator receptor (PPAR) agonist,aramchol, a caspase inhibitor, a galectin 3 inhibitor, an FGF19 agonist,an FGF21 agonist, a LTD4 receptor antagonist, a niacin analog, an ASBTinhibitor, an apoptosis signal regulating kinase 1 (ASK1) inhibitor, anangiotensin converting enzyme (ACE) inhibitor, an angiotensin receptorblocker (ARB), a chemokine receptor inhibitor, a thiozolidinedione, aGLP-1 analog, a biguanide, or an NSAID. In some embodiments, theubenimex is administered at a daily dosage of 1000 mg or less. In someembodiments, the ubenimex and the secondary pharmaceutical agent areadministered for at least 4 weeks.

In another aspect, the disclosure also provides for the manufacture of amedicament for the treatment of NASH and/or for prevention of theprogression of NAFLD to NASH, wherein the active ingredient in themedicament is ubenimex. In some embodiments, the medicament is apharmaceutical composition comprising ubenimex and at least onepharmaceutically acceptable carrier. In various embodiments, themedicament is formulated for oral administration, including immediaterelease and sustained release pharmaceutical formulations. Thedisclosure also provides for the manufacture of unit dosage forms of themedicament useful in treating patients and pharmaceutical packs and kitscomprising one or more containers with a solid or liquid formulation ofubenimex as described herein.

In some embodiments, pharmaceutical preparations comprising ubenimex forthe treatment of non-alcoholic steatohepatitis are provided. In someembodiments, the pharmaceutical preparation is in the form of a tablet,capsule, or pill suitable for oral administration. In some embodiments,the pharmaceutical preparation comprises ubenimex in an amount rangingfrom about 5 mg to about 450 mg.

In another aspect, methods of treating non-alcoholic steatohepatitis(NASH) are provided, comprising administering to a subject in need oftreatment a therapeutically effective amount of ubenimex. In someembodiments, the subject has early-stage or middle-stage NASH. In someembodiments, the ubenimex is administered at a total daily dose in therange of 5 mg to 1000 mg. In some embodiments, the ubenimex isadministered at a daily dose of about 5 mg to about 450 mg. In someembodiments, the ubenimex is administered at a daily dose of about 20 mgto about 450 mg QD. In some embodiments, the ubenimex is administered ata daily dose of about 10 mg to about 200 mg BID. In some embodiments,the ubenimex is administered at a daily dose of about 10 mg to about 150mg BID. In some embodiments, the ubenimex is administered for at least 4weeks. In some embodiments, the ubenimex is administered for at least 12weeks. In some embodiments, treatment results in a reduction in plasmaCK-18 levels in the subject. In some embodiments, treatment results in areduction in hepatocyte ballooning in the subject.

In some embodiments, the method of treating NASH comprises administeringubenimex in combination with a second therapeutic agent. In someembodiments, the second therapeutic agent is a farnesoid X receptor(FXR) agonist, a peroxisome proliferator-activator receptor (PPAR)agonist, aramchol, a caspase inhibitor, a galectin 3 inhibitor, amitogen-activated protein kinase 5 (MAPK5) inhibitor, a fibroblastgrowth factor 19 (FGF19) agonist, a FGF21 agonist, a leukotriene D4(LTD4) receptor antagonist, a niacin analog, an apical sodium bile acidcotransporter (ASBT) inhibitor, an apoptosis signal regulating kinase 1(ASK1) inhibitor, an angiotensin converting enzyme (ACE) inhibitor, anangiotensin receptor blocker, a chemokine receptor inhibitor, athiozolidinedione, a GLP-1 analog, a biguanide, or a non-steroidalanti-inflammatory drug (NSAID).

In another aspect, methods of delaying or preventing the progression ofnon-alcoholic fatty liver disease (NAFLD) to NASH in a subject havingNAFLD are provided. In some embodiments, the method comprisesadministering to the subject a therapeutically effective amount ofubenimex. In some embodiments, treatment results in a reduction inhepatocyte ballooning in the subject. In some embodiments, the ubenimexis administered at a total daily dose in the range of 5 mg to 1000 mg.In some embodiments, the ubenimex is administered at a daily dose ofabout 5 mg to about 450 mg. In some embodiments, the ubenimex isadministered at a daily dose of about 20 mg to about 450 mg QD. In someembodiments, the ubenimex is administered at a daily dose of about 10 mgto about 200 mg BID. In some embodiments, the ubenimex is administeredat a daily dose of about 10 mg to about 150 mg BID. In some embodiments,the ubenimex is administered for at least 4 weeks. In some embodiments,the ubenimex is administered for at least 12 weeks.

In some embodiments, the method of delaying or preventing theprogression of NAFLD to NASH comprises administering ubenimex incombination with a second therapeutic agent. In some embodiments, thesecond therapeutic agent is a farnesoid X receptor (FXR) agonist, aperoxisome proliferator-activator receptor (PPAR) agonist, aramchol, acaspase inhibitor, a galectin 3 inhibitor, a mitogen-activated proteinkinase 5 (MAPK5) inhibitor, a fibroblast growth factor 19 (FGF19)agonist, a FGF21 agonist, a leukotriene D4 (LTD4) receptor antagonist, aniacin analog, an apical sodium bile acid cotransporter (ASBT)inhibitor, an apoptosis signal regulating kinase 1 (ASK1) inhibitor, anangiotensin converting enzyme (ACE) inhibitor, an angiotensin receptorblocker, chemokine receptor inhibitor, a thiozolidinedione, a GLP-1analog, a biguanide, or a non-steroidal anti-inflammatory drug (NSAID).

In still another aspect, methods of decreasing hepatocyte ballooning ina subject having NAFLD and/or NASH are provided. In some embodiments,the method comprises administering to the subject a therapeuticallyeffective amount of ubenimex for at least 4 weeks. In some embodiments,the method comprises administering to the subject a therapeuticallyeffective amount of ubenimex for at least 12 weeks. In some embodiments,the subject has NAFLD. In some embodiments, the subject has NASH.

In yet another aspect, methods of decreasing inflammation in a subjecthaving NAFLD and/or NASH are provided. In some embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of ubenimex for at least 24 weeks. In some embodiments, themethod comprises administering to the subject a therapeuticallyeffective amount of ubenimex for at least 48 weeks. In some embodiments,the subject has NAFLD. In some embodiments, the subject has NASH.

In yet another aspect, methods of decreasing fibrosis in a subjecthaving NASH are provided. In some embodiments, the method comprisesadministering to the subject a therapeutically effective amount ofubenimex for at least 24 weeks. In some embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of ubenimex for at least 48 weeks.

In yet another aspect, pharmaceutical packages comprising unit dosageforms of ubenimex and further comprising unit dosage forms of a secondtherapeutic agent are provided. In some embodiments, the secondtherapeutic agent is a FXR agonist, a PPAR agonist, aramchol, a caspaseinhibitor, a galectin 3 inhibitor, a MAPK5 inhibitor, a FGF19 agonist, aFGF21 agonist, a LTD4 receptor antagonist, a niacin analog, an ASBTinhibitor, an ASK1 inhibitor, an ACE inhibitor, an angiotensin receptorblocker, a chemokine receptor inhibitor, a thiozolidinedione, a GLP-1analog, a biguanide, or an NSAID. In some embodiments, the secondtherapeutic agent is not a chemotherapy agent. In some embodiments, eachunit dosage form of ubenimex comprises ubenimex in an amount from 5 mgto 1000 mg. In some embodiments, each unit dosage form of ubenimexcomprises ubenimex in an amount from about 5 mg to about 450 mg. In someembodiments, the ubenimex is formulated for immediate release. In someembodiments, the ubenimex is formulated for controlled release. In someembodiments, the ubenimex is in the form of a tablet, a capsule, or apill.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D. NAFLD activity score and component scores in mice treatedwith vehicle, ubenimex QD for 5 weeks, ubenimex BID for 5 weeks,ubenimex BID for 2 weeks, or telmisartan for 5 weeks. (A) NAFLD activityscores for the five groups. (B) Steatosis component scores for the fivegroups. (C) Inflammation component scores for the five groups. (D)Hepatocyte ballooning component scores for the five groups.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver diseasethat ranges from simple steatosis to non-alcoholic steatohepatitis(NASH), fibrosis, and cirrhosis. The key histological features of NASHinclude steatosis, hepatocyte ballooning, and lobular inflammation, andfibrosis is also typically observed. Takahashi et al., WorldGastroenterol, 2014, 20:15539-15548.

As described in the Examples section below, it has been found that in ananimal model of NASH, ubenimex demonstrated efficacy in slowing andpreventing hepatocyte ballooning and reduced steatosis and lobularinflammation after only three weeks of dosing. Accordingly, in oneaspect, methods of treating one or more symptoms of NAFLD and/or NASH,such as hepatocyte ballooning, steatosis, and lobular inflammation, areprovided. Furthermore, Example 1 describes that plasma CK-18 levelsdeclined significantly relative to those measured in control animals.Thus, in some embodiments, administration of ubenimex as describedherein decreases ballooning and measurably lowers plasma and/or liverCK-18 levels in as few as 3 to 12 weeks after treatment initiation. Insome embodiments, continued daily administration of ubenimex isefficacious in decreasing inflammation and fibrosis in NASH within 24 to48 weeks after treatment initiation. In some embodiments, treatment withubenimex results in an improvement in one or more parameters such asimproved ALT enzyme levels, decreased inflammation, decreased steatosis,reduced severity of NASH symptoms, reduced levels of NASH biomarkerssuch as CK-18, or the slowing, stopping, or reversing of liver fibrosis.

II. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. In thisspecification and in the claims that follow, reference will be made to anumber of terms, which shall be defined to have the definitions setforth below. Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. The singularforms also include the plural unless the context clearly dictatesotherwise. Thus, the singular forms “a,” and “the” include pluralreferents unless the context clearly dictates otherwise.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 1.0, as appropriate. It is tobe understood, although not always explicitly stated that all numericaldesignations are preceded by the term “about.”

Acronyms. The following acronyms are used throughout the specificationand defined as follows: NASH: non-alcoholic steatohepatitis; NAFLD:non-alcoholic fatty liver disease; QD: one a day; BID: twice a day; TID:three times a day.

As used herein, “active agent” refers to a compound or drug that exertsa preventative or therapeutic effect on a disease or condition. As usedherein, “active agent” can refer to either a single active agent or to acombination of two or more different active agents.

The terms “administer” and “administration” refer to a method ofdelivering a compound, a composition, or an agent to the desired site ofbiological action. These methods include, but are not limited to, oraldelivery, intravenous delivery, parenteral delivery, intramusculardelivery, intraperitoneal delivery, or subcutaneous delivery.

As used herein, “capsules” are unit dosage forms (e.g., for oraladministration) in which the active agent-containing composition may beencapsulated in the form of a liquid or solid (including particulatessuch as granules, beads, powders or pellets). Suitable capsules may beeither hard or soft, and are typically made of gelatin, starch, or acellulosic material. In some embodiments, the capsules are gelatincapsules.

The term “compound” refers to a molecule and encompasses not only thespecified molecular entity but, if the compound is an active agent ordrug, also its pharmaceutically acceptable, pharmacologically activeanalogs, including, but not limited to, active metabolites, amides,conjugates, esters, hydrates, polymorphs, prodrugs, salts, solvates, andother such derivatives, analogs, including deuterated analogs andanalogs containing radioactive atoms or other labeling moieties, andrelated compounds.

The term “comprising” is intended to mean that the compounds,compositions and methods include the recited elements, but not excludingothers. “Consisting essentially of,” when used to define compounds,compositions and methods, means excluding other elements that wouldmaterially affect the basic and novel characteristics of the claimedinvention. “Consisting of” means excluding any element, step, oringredient not specified in the claim. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “CK-18” refers to cytokeratin-18 fragment, which has beenidentified as a noninvasive biomarker for NASH in that it is markedlyincreased in patients with NASH as TO determined by histology and higherblood plasma levels of the fragment correlate with the odds of havingfibrosis on liver biopsy. See, Feldstein et al., Hepatology, 2009,50:1072-8, incorporated by reference herein.

The term “dosage form” refers to a form of a pharmaceutical compositionfor administration to a subject (e.g., a human or non-human animalhaving a disease or condition to be treated). “Dose” refers to an amountof active agent. “Unit dosage form” refers to a dosage form thatcontains a fixed amount of active agent. For example, a single tablet orcapsule is a unit dosage form. In some embodiments, multiple unit dosageforms are administered to provide a therapeutically effective dose.

The term “oral unit dosage form,” as used herein, refers to a unitdosage form that is intended to be orally administered.

The terms “effective amount” and “therapeutically effective amount”refer to an amount of an active agent being administered that will treatto some extent a disease, disorder, or condition, e.g., relieve one ormore of the symptoms of the disease being treated (e.g., NASH), and/orthat amount that will prevent, to some extent, one or more of thesymptoms of the disease that the subject being treated has or is at riskof developing.

For example, for a given parameter, a therapeutically effective amountwill show an increase or decrease of therapeutic effect of at least 5%,10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol.

An “excipient” or “carrier,” as used herein, refers to a biologicallyinactive substance used in combination with an active agent(s) of theformulation. An excipient can be used, for example, as a solubilizingagent, a stabilizing agent, a diluent, an inert carrier, a preservative,a binder, a disintegrant, a coating agent, a flavoring agent, or acoloring agent. A wide variety of pharmaceutically acceptableexcipients, such as vehicles, adjuvants, carriers or diluents, andauxiliary substances, such as pH adjusting and buffering agents,tonicity adjusting agents, stabilizers, wetting agents and the like, areknown in the art. Pharmaceutically acceptable excipients have been amplydescribed in a variety of publications, including, for example, A.Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20thedition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Formsand Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. PharmaceuticalAssoc.

The term “parenteral administration,” as used herein, refers to anon-oral means of administration, and includes subcutaneous,intravenous, and intramuscular routes of administration.

The term “pharmaceutical composition” refers to a composition that issuitable for administration to a subject. In general a “pharmaceuticalcomposition” is sterile, and preferably free of contaminants that arecapable of eliciting an undesirable response within the subject (e.g.,the compound(s) in the pharmaceutical composition is pharmaceuticalgrade). Pharmaceutical compositions can be designed for administrationto subjects or patients in need thereof via a number of different routesof administration including oral, intravenous, buccal, rectal,parenteral, intraperitoneal, intradermal, intratracheal, intramuscular,subcutaneous, inhalational, and the like.

The term “pharmaceutically acceptable,” as used with reference to acompound or component, means that the compound or component is generallysafe, non-toxic, and not biologically undesirable. When the term“pharmaceutically acceptable” is used herein to refer to apharmaceutical carrier or excipient, it is implied that the carrier orexcipient has met the required standards of toxicological andmanufacturing testing or that it is included on the Inactive IngredientGuide prepared by the U.S. Food and Drug Administration.

The term “pharmaceutically acceptable salt” refers to a derivative of anactive agent produced by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. Pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids.Pharmaceutically acceptable salts include those formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like.Pharmaceutically acceptable salts include solvent addition forms(solvates) or crystal forms (polymorphs) as defined herein, of the samesalt.

The terms “prevention,” “preventing,” and “prevent” mean avoiding theonset of a clinically evident disease progression altogether or slowingthe onset of a pre-clinically evident stage of a disease in individualsat risk. Prevention includes prophylactic treatment of those at risk ofdeveloping a disease.

The term “sign,” as used herein, means an indication of disease andincludes conditions that can be observed by a doctor, nurse, or otherhealth care professional.

The terms “subject” and “patient” interchangeably refer to a human ornon-human animal (e.g., mammal) suitable for treatment with an activeagent. A subject in need thereof may have a disease (e.g., NASH) or maybe at an increased risk, relative to the general population, ofdeveloping a disease (e.g., NASH). In some embodiments, a subject hasbeen diagnosed with a disease (e.g., NASH).

The term “symptom” means a sign or other indication of disease, illness,or injury. Symptoms may be felt or noticed by the individualexperiencing them or by others, including by non-health-careprofessionals.

The terms “treatment,” “treating,” and “treat” refer to any indicia ofsuccess in the treatment or amelioration of an injury, disease, orcondition, including any objective or subjective parameter such asabatement, remission, improvement in patient survival, increase insurvival time or rate, diminishing of symptoms or making the injury,disease, or condition more tolerable to the patient, slowing in the rateof degeneration or decline, or improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment, or to the same patient prior to treatment or at a differenttime during treatment.

“Ubenimex” refers to(2S)-2-[[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]amino]-4-methylpentanoicacid, which is also known asN-[(2S,3R)-3-Amino-2-hydorxy-4-phenylbutyryl-L-leucine, the structure ofwhich is shown below:

Ubenimex is a zwitterionic molecule that has a solubility of 1.27 mg/mLin water and has a melting point of approximately 251° C. Ubenimex isdescribed in U.S. Pat. Nos. 4,029,547 and 4,052,449, incorporated byreference herein. Reference herein to ubenimex also includes a referenceto a pharmaceutically acceptable salt of ubenimex unless otherwiseindicated or clear from context. In some embodiments, ubenimex is in theform of ubenimex hydrochloride. Ubenimex and pharmaceutically acceptablesalts thereof are commercially available (e.g., Tocris Bioscience).Ubenimex can also be prepared according to methods known in the art.See, e.g., U.S. Pat. No. 4,029,547, incorporated by reference herein.

All percentages are % w/w, unless otherwise specified. Unless otherwiseindicated, “% weight” is percent weight of the specified componentcompared to total weight of the unit dosage (e.g., tablet or capsule).It will be appreciated that due to rounding or practical limits onquantitative measurements, reference to a quantity of API or excipientin a dosage form can include some variation, such as ±0.10% or ±0.5%.

III. Methods of Treating NAFLD and NASH

In one aspect, the present disclosure provides a therapy for thetreatment of NASH that comprises administering to a patient in need oftreatment a therapeutically efficacious dose of ubenimex. In someembodiments, the patient in need of treatment is a patient who has beendiagnosed with NASH. In some embodiments, treatment with ubenimex asdescribed herein slows, stops, or reverses NASH disease progression.

In another aspect, the present disclosure provides a therapy for theprevention of NASH or for slowing the progression of NAFLD to NASH byadministering to a patient in need of treatment a therapeuticallyefficacious dose of ubenimex. In some embodiments, the patient in needof treatment is a patient who has been diagnosed with NAFLD.

Patient Population

Patients likely to benefit from the therapies of the present disclosurecan be readily identified by a variety of means discussed herein orknown to those of skill in the art. In addition, methods for determiningwhether a patient is responding to this therapy are also provided. Insome embodiments, abdominal imaging tests, including ultrasoundexamination, computerized tomography (CT), and/or magnetic resonanceimaging (MRI) can be used to diagnose patients with the disease, e.g.evaluate whether the disease is present and its severity. Such anon-invasive diagnosis can be more definitively confirmed by liverbiopsy, if desired. In some embodiments, one or more biomarkers is usedto diagnose NAFLD or NASH. In some embodiments, a patient to be treatedin accordance with the present disclosure has received a primarydiagnosis of NASH or NAFLD and is not being treated with ubenimex forany other condition for which it is currently indicated or in clinicaldevelopment (e.g., certain cancer patients, PAH indication, orlymphedema indication). NAFLD is characterized by significant lipiddeposition in hepatocytes and is typically defined as either excessivefat accumulation in the liver (with more than 5% of hepatocytescontaining visible intracellular triglycerides) or steatosis affectingat least 5% of the liver volume or weight. El-Kader et al., World J.Hepatol, 2015, 7:846-858. In some embodiments, a patient to be treatedhas NAFLD. Some patients also exhibit abnormal liver function, e.g., asdetermined by the presence of elevated serum aspartate aminotransferase(ALT), gamma glutamyl transpeptidase, or alkaline phosphatase levels. Insome embodiments, a patient to be treated has NAFLD and further has anelevated ALT level, an elevated gamma glutamyl transpeptidase, or anelevated alkaline phosphatase level (e.g., a level that is about 1.5- to4-fold above the upper limit of normal). In some embodiments, a patientto be treated has NAFLD and has an ALT level, gamma glutamyltranspeptidase level, or alkaline phosphatase level that is within theupper limit of normal.

In some embodiments, NAFLD is diagnosed using an imaging test. In someembodiments, NAFLD is diagnosed using a scoring system such as but notlimited to fatty liver index (in which a score >60 indicates a high riskfor NAFLD), NAFLD liver fat score, NAFLD activity score, or hepaticsteatosis index. In some embodiments, NAFLD is diagnosed using a NAFLDactivity score (NAS), which provides a composite score based on thedegree of steatosis (0-3), lobular inflammation (0-3), and hepatocyteballooning (0-2). See, Kleiner et al., Hepatology, 2005, 41:1313-1321;Bugianesi et al., J Hepatology, 2016, 65:643-644.

NASH has been classified pathologically into type 1 and type 2 forms, ofwhich the type 1 form is more commonly found in adult patients, whilethe type 2 form is more commonly found in children. Type 1 NASH istypically characterized by steatosis, hepatocyte ballooning, andperisinusoidal fibrosis. Type 2 NASH is typically characterized bysteatosis, portal inflammation, and portal fibrosis. See, e.g.,Schwimmer et al., Hepatology, 2005, 42:641-649. Further progression ofNASH can lead to severe fibrosis, cirrhosis, and end-stage liverdisease. In some embodiments, a patient to be treated has Type 1 NASH.In some embodiments, a patient to be treated has Type 2 NASH. In someembodiments, a patient to be treated has early-stage NASH. In someembodiments, a patient to be treated has middle-stage NASH. In someembodiments, a patient to be treated has late-stage NASH (e.g., hassevere fibrosis and/or cirrhosis of the liver).

In some embodiments, NASH is diagnosed using an imaging test. In someembodiments, NASH is diagnosed using a scoring system such as but notlimited to NAFLD activity score (e.g., a score of ≥5) or a steatosis,activity, and fibrosis (SAF) score, or a NAFLD fibrosis score; a serumbiomarker (e.g., cytokeratin-18); or a combination thereof. See, Bedossaet al., Hepatology, 2012, 56:1751-1759; Arab et al., GastroenterolHepatol, 2017, 40:388-394. In some embodiments, fibrosis is detectedand/or measured using elastography (e.g., Fibroscan®).

In some embodiments, a patient to be treated is identified by use of oneor more biomarkers such as CK-18. CK-18 levels, whether measured byimmunohistochemistry, histology from liver biopsies, or via measurementof plasma levels in patients or individuals suspected of being at riskfor the disease, will typically be elevated, relative to the levelsmeasured in healthy individuals, in subjects in need of treatment. Whilethe invention is not to be limited to a particular or any proposedmechanism of action, decreased CK-18 levels in NASH patients would beexpected to correlate with decreased liver cell apoptosis. Accordingly,patients with NAFLD or NASH who are treated with ubenimex in accordancewith the present disclosure should benefit from decreased liver cellapoptosis, relative to receiving no treatment or standard of care.

In some embodiments, a patient to be treated is a human adult. In someembodiments, a patient to be treated is a human child under 18 years ofage (e.g., from age 2 to age 17).

In some embodiments, a patient to be treated does not have cancer. Insome embodiments, a patient to be treated does not have acutenon-lymphocytic leukemia. In some embodiments, a patient to be treateddoes not have lymphedema. In some embodiments, a patient to be treateddoes not have pulmonary arterial hypertension.

Dosage Regimen

In some embodiments, ubenimex is administered at a total daily dose thatis about 1000 mg or less (e.g., less than 900 mg, less than 800 mg, lessthan 750 mg, less than 700 mg, less than 600 mg, or less than 500 mg).As used herein, the dose amount refers to the amount of activeingredient administered per dose, not the amount of the pharmaceuticalformulation. In some embodiments, ubenimex is administered at a totaldaily dose in the range of about 5 mg to about 450 mg, e.g., from about10 mg to about 450 mg, from about 20 mg to about 450 mg, from about 30mg to about 450 mg, from about 10 mg to about 350 mg, from about 20 mgto about 350 mg, from about 30 mg to about 350 mg, from about 10 mg toabout 250 mg, from about 20 mg to about 250 mg, or from about 30 mg toabout 250 mg. This daily dose may be administered all at once: one timedaily, although in some embodiments the once daily dose (QDadministration) will be at least 30 mg or more. For BID administration,in some embodiments the daily dose will be 10-450 mg, e.g. 5-225 mg twotimes daily, although in some embodiments the dose will be at least 15mg or more for BID administration. For TID administration, in someembodiments the daily dose will be 15-450 mg, e.g. 5-150 mg three timesdaily, although in some embodiments the dose will be at least 10 mg ormore for TID administration. In some embodiments, ubenimex isadministered at a daily dose of at least 20 mg, at least 30 mg, at least40 mg, or at least 50 mg. In some embodiments, ubenimex is administeredat a daily dose of about 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440mg, or 450 mg.

In some embodiments, ubenimex is administered at a dosage of about 10 mgto 450 mg QD, e.g., about 15 mg to 400 mg QD, about 20 mg to about 350mg QD, about 20 mg to about 300 mg QD, about 25 mg to about 250 mg QD,or about 25 mg to about 200 mg QD. In some embodiments, ubenimex isadministered at dosage of about 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg,160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg,250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg,340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg,430 mg, 440 mg, or 450 mg QD.

In some embodiments, ubenimex is administered at a dosage of about 10 mgto 200 mg BID, e.g., about 20 mg to about 150 mg BID, about 20 mg toabout 100 mg BID, or about 25 mg to about 150 mg BID. In someembodiments, ubenimex is administered at dosage of about 20 mg, 25 mg,30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg,130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg BID.

In some embodiments, ubenimex is administered at a dosage of about 10 mgto 150 mg TID, e.g., about 20 mg to about 150 mg TID, about 20 mg toabout 100 mg TID, or about 25 mg to about 150 mg TID. In someembodiments, ubenimex is administered at dosage of about 20 mg, 25 mg,30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg,130 mg, 140 mg, or 150 mg TID.

Formulations and unit dosage forms of ubenimex are further disclosed inSection IV below. In some embodiments, ubenimex is administered orally.In some embodiments, ubenimex is administered orally in atherapeutically effective dose at least once and no more than thricedaily on consecutive days for at least a week and typically longer.

In some embodiments, ubenimex is administered alone (i.e., not incombination with another medication) for part or all of the treatmentperiod. In some embodiments, ubenimex is administered in combinationwith one or more other drugs, such as but not limited to a farnesoid Xreceptor (FXR) agonist (including but not limiting to obeticholic acid);a peroxisome proliferator-activator receptor (PPAR) agonist, e.g., aPPAR-alpha agonist, a PPAR-beta/delta agonist, or a PPAR-gamma agonist(including but not limited to elafibranor); aramchol; a caspaseinhibitor (including but not limited to emricasan); a galectin 3inhibitor (including but not limited to GR-MD-02, GalectinTherapeutics); a MAPK5 inhibitor (including but not limited to GS-4997,Gilead Sciences); an FGF21 agonist (including but not limited toBMS-986036, Bristol-Myers Squibb); a LTD4 receptor antagonist (includingbut not limited to tipelukast); a niacin analog (including but notlimited to ARI 3037MO, Arisaph Pharmaceuticals); an ASBT inhibitor(including but not limited to volixibat); an apoptosis signal regulatingkinase 1. (ASK1) inhibitor; an ACE inhibitor or ARBs (includingtelmisartan); a CCR2 and/or CCR5 inhibitor (including cenicriviroc); athiazolidinedione (including rosiglitazone and pioglitazone); a GLP-1analog (including but not limiting to semaglutide, liraglutide); or abiguanide such as metformin. Without being bound to a particular theory,it is believe that such treatments in accordance with the disclosure canimprove insulin sensitivity in adipose tissue by activating PPAR andgenerate biochemical and histological improvements in NASH. Combinationtherapy with ubenimex and a second therapeutic agent is also disclosedin Section V below.

In some embodiments, ubenimex is administered as a pharmaceuticalformulation suitable for oral, intranasal, rectal, topical,intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal,transdermal, or intrathecal administration. In some embodiments,pharmaceutical formulations for use according to the present disclosureare prepared for oral administration and in an immediate release formsuitable for QD, BID, or TID administration, and the dosage regimen isselected within the ranges provided herein in accordance with a varietyof factors including type, species, age, weight, sex and medicalcondition of the patient; the severity of the condition to be treated;the route of administration; the renal and hepatic function of thepatient; and the particular formulation employed. An ordinarily skilledphysician or veterinarian can readily determine and prescribe theeffective amount of the drug required to prevent, counter or arrest theprogress of the condition in view of the teachings herein.

Duration of Treatment, Treatment Endpoints, and Monitoring Efficacy

In some embodiments, treatment with ubenimex (and optionally a secondtherapeutic agent) is administered for a predetermined time, anindefinite time, or until an endpoint is reached. In some embodiments,treatment is for at least 30 days or one month, at least 60 days or twomonths, at least 90 days or three months, at least 120 days or fourmonths, at least 150 days or five months, or at least 180 days or sixmonths. In some embodiments, treatment is continued for at least sixmonths to one year. In some embodiments, ubenimex is administered (e.g.,by consecutive daily administration of ubenimex as described herein) forat least a month, for at least 3 months, or for at least 6 months to atleast a year. In other embodiments, treatment is continued for the restof the patient's life or until administration is no longer effective inproviding a meaningful therapeutic benefit. In some embodiments,treatment is administered on a continuous daily basis. In someembodiments, treatment is administered on a near continuous daily basis(e.g., ubenimex treatment is administered to a patient daily but thepatient may occasionally miss a day of treatment).

In some embodiments, generally continuous (or near continuous) dailydosing is continued until treatment appears to no longer have abeneficial effect or until unacceptable side effects appear. Manypatients will take the medication for at least a week, at least a month,and at least a year or longer. In some instances, a patient will takethe medication from approximately 6 months to approximately 1 year. Insome instances, a patient will take the medication for greater than 1year. Many patients will take the medication for the rest of theirlives.

In some embodiments, treatment according to the methods described hereinresults in an improvement in one or more parameters such as, but notlimited to, an improvement in NAS (ballooning and inflammation) and/orfibrosis; an improvement in SAF (steatosis, activity, and fibrosis)score; complete resolution of steatohepatitis; no worsening of fibrosis;an improvement in fibrosis without a worsening of steatohepatitis; or anincreased time to disease progression as measured by histopathologicassessment of progression to cirrhosis, death, liver transplant,hepatocellular carcinoma, and decompensation events such as hepaticencephalopathy, variceal bleeding requiring hospitalization, ascitesrequiring intervention, and spontaneous bacteria peritonitis. In someembodiments, treatment according to the methods described herein resultsin an improvement (i.e., a reduction) in hepatocyte ballooning. In someembodiments, hepatocyte ballooning is visualized using hematoxylin andeosin straining.

In some embodiments, treatment according to the methods described hereinresults in an improvement in one or more biomarkers of NAFLD or NASH,such as but not limited to markers of apoptosis (e.g., cytokeratin(CK-18) fragments), adipokines (e.g., adiponectin, leptin, resistin, orvisfatin), inflammatory markers (e.g., TNF-α, IL-6, chemo-attractantprotein-1, or high sensitivity C-reactive protein). See, e.g., Neuman etal., Can J Gastroenterol Hepatol, 2014, 28:607-618; Castera et al., NatRev Gastroenterol Hepatol., 2013, 10:666-675. In some embodiments,biomarker values are measured using a sample that comprises a fluid,e.g., blood, plasma, serum, urine, or cerebrospinal fluid. In someembodiments, biomarker values are measured using a sample that comprisescells and/or tissues, e.g., hepatocytes or liver tissue. In someembodiments, treatment results in an improvement in the biomarker CK-18.In some embodiments, treatment results in a reduction in plasma CK-18levels in the subject.

In some embodiments, a patient is monitored during the course ofubenimex therapy using a diagnostic test as described herein (e.g.,using abdominal imaging tests). In some embodiments, the method furthercomprises continuing a course of therapy (e.g., a dosage of ubenimex asdescribed herein). In some embodiments, the method further comprisestapering, reducing, or stopping the administered amount of ubenimex ifthe diagnosis warrants, e.g. when a cure is effected, a lower doseappears to be safer or equally efficacious as a higher dose, or nocontinuing therapeutic effect is expected. In some embodiments, themethods can comprise increasing the administered amount of ubenimex ifit is determined not to be efficacious, as well as stopping therapy ifit is determined dose escalation or continued dosing at any dose isunlikely to be efficacious.

In some embodiments where the patient is undergoing treatment inaccordance with the present disclosure, indications of NASH by abdominalimaging, ultrasound examination, magnetic resonance imaging, CT scan,and/or biopsy may be less than those measured in the patient prior totreatment, which is indicative that the patient is responding positivelyto the therapy. In cases where the patient is responding positively to atherapy of the present disclosure, the therapy is continued until thepresence of the condition is reduced to a level comparable to a normalcontrol level. Optionally, the therapy is continued to maintainalleviation of NASH symptoms. Alternatively, the therapy is continueduntil a desired level of steatosis is achieved in the patient (includingthe absence of steatosis). Treatment may be continued for so long as itis determined to be efficacious using assessment by abdominal imaging,ultrasound examination, magnetic resonance imaging, CT scan, and/orbiopsy. The treatment may be determined to be efficacious throughmeasured improvement in one or more of steatosis, ballooning, andnecroinflammation. In one embodiment, the treatment is determined to beefficacious through measured improvement indicated by induced reductionin ballooning. In one embodiment, the treatment is determined to beefficacious through measured improvement indicated by a reduction ininflammation. In one embodiment, the treatment is determined to beefficacious through measured improvement indicated by at least one ofreduced serum ALT levels, improved insulin sensitivity, reducedsteatosis, reduced inflammation, reduced fibrosis, and increased PPAR(e.g., PPAR-alpha, PPAR-beta/delta, or PPAR-gamma) activation. In oneembodiment, the treatment is determined to be efficacious throughmeasured improvement indicated by induced regression or reversal offibrosis and/or cirrhosis.

In some embodiments, treatment results in an improvement in one or moreparameters (e.g., a reduction in NAS or SAF score, a reduction inhepatocyte ballooning, a reduction in fibrosis, or a reduction in CK-18levels) of at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90% ascompared to a control value. In some embodiments, treatment results inan improvement in one or more parameters of at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold ascompared to a control value. In some embodiments, the control value is abaseline value for the subject that is determined prior to the onset oftreatment.

In some embodiments, the present disclosure provides methods ofdetermining efficacy of a NASH treatment in a subject in need thereof by(a) measuring the level and severity of NASH via abdominal imaging,ultrasound examination, magnetic resonance imaging, CT scan, and/orliver biopsy in a subject in need thereof, where the level and severityof NASH is measured after treatment has started, (b) comparing the leveland severity of NASH as measured in step (a) to a baseline level andseverity of NASH, where the baseline level and severity is measured inthe same subject before treatment is begun, and (c) determining theefficacy of the NASH treatment based on the comparison step.

Furthermore, in some embodiments the present disclosure provides methodsof determining efficacy of a NASH treatment in a subject in need thereofby (a) measuring the level and severity of NASH in a subject in needthereof after treatment has begun, (b) comparing the level and severityof the NASH to a reference value, where the reference value representsan average value determined from a population of patients suffering fromNASH, and (c) determining the efficacy of the NASH treatment based onthe comparison step. In some embodiments, efficacy of therapy isdetermined by liver biopsy and analysis to evaluate NAFLD Activity Score(NAS) and fibrosis; the transjugular liver biopsy method can be employedfor this purpose. Suitable patients include patients with biopsy provenNASH, patients at high risk for NASH, patients with a NAS greater thanor equal to 4, NASH patients with liver fibrosis, and NASH patients withliver fibrosis of stage 2 or greater.

In some embodiments, patients responding to therapy in accordance withthe invention are expected to show at least a slowing of any increase inCK-18 levels as therapy continues. In some embodiments, those patientsresponding most favorably to therapy will have CK-18 levels thatstabilize and decline over time as full therapeutic benefit is realized.Thus, in some embodiments, the present disclosure provides methods ofdetermining efficacy of a NASH treatment in a subject in need thereof by(a) measuring the level and severity of NASH via measuring the level ofthe biomarker CK-18 in a sample from the sample from the subject (e.g.,a blood, plasma, or tissue sample), wherein the level and severity ofNASH is measured after treatment has started, (b) comparing the leveland severity of NASH measured in (a) to a baseline level and severity ofNASH in the subject that is measured in the same subject beforetreatment is begun, and (c) determining the efficacy of the NASHtreatment based on the comparison step; wherein a plateau or decrease inCK-18 levels is indicative of efficacy of the NASH treatment.

IV. Compositions, Unit Dosage Forms, and Kits Comprising Ubenimex

In another aspect, compositions, unit dosage forms, pharmaceuticalpackages, and kits comprising ubenimex for use in the methods describedherein are provided. In some embodiments, the formulations, unit dosageforms, pharmaceutical packages, and/or kits are for use in treatingNASH. In some embodiments, the formulations, unit dosage forms,pharmaceutical packages, and/or kits are for use in delaying orpreventing the progression of NAFLD to NASH in a subject having NAFLD.

In some embodiments, the compositions, unit dosage forms, pharmaceuticalpackages, and/or kits comprise ubenimex or a pharmaceutically acceptablesalt thereof. In some embodiments, the composition, unit dosage form,pharmaceutical package, or kit comprises ubenimex or ubenimexhydrochloride. In some embodiments, a composition or unit dosage formcomprises ubenimex or a pharmaceutically acceptable salt thereof in anamount from about 10% to 50%, or from about 20% to about 40%, or about30% to about 35%, or from about 15% to about 25% by weight of the totalcomposition. In some embodiments, ubenimex is provided as an amorphoussolid dispersion.

In some embodiments, ubenimex is formulated in a pharmaceuticalcomposition comprising one or more pharmaceutically acceptableexcipients. In some embodiments, an excipient comprises a solubilizingagent, a stabilizing agent, a diluent, an inert carrier, a preservative,a binder, a disintegrant, a coating agent, a flavoring agent, or acoloring agent. Suitable pharmaceutical compositions, formulations, andunit dosage forms may be prepared using conventional methods known tothose in the field of pharmaceutical formulation and described in thepertinent texts and literature, e.g., in Remington: The Science andPractice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995).Typically, pharmaceutical formulations of the disclosure compriseubenimex and one or more pharmaceutically acceptable (approved by astate or federal regulatory agency for use in humans, or is listed inthe U.S. Pharmacopia, the European Pharmacopia) excipients or carriers.

In some embodiments, at least one excipient is chosen to provide one ormore beneficial physical properties to the formulation, such asincreased stability and/or solubility of the active agent(s). Examplesof suitable excipients include certain inert proteins such as albumins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asaspartic acid (which may alternatively be referred to as aspartate),glutamic acid (which may alternatively be referred to as glutamate),lysine, arginine, glycine, and histidine; fatty acids and phospholipidssuch as alkyl sulfonates and caprylate; surfactants such as sodiumdodecyl sulphate and polysorbate; nonionic surfactants such as TWEEN®,PLURONIC®, or polyethylene glycol (PEG); carbohydrates such as glucose,sucrose, mannose, maltose, trehalose, and dextrins, includingcyclodextrins; polyols such as mannitol and sorbitol; chelating agentssuch as EDTA; and salt-forming counter-ions such as sodium.

In some embodiments, ubenimex is formulated as a liquid pharmaceuticalcomposition, such as a composition comprising ubenimex in suspension,solution, or emulsion in an oily or aqueous vehicle. In someembodiments, the liquid composition further comprises one or moreformulatory agents such as suspending, stabilizing, and/or dispersingagents. In some embodiments, solutions or suspensions used for thedelivery of a drug in liquid formulation can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,polysorbate, tocopherol polyethylene glycol succinate (TPGS), or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide.These preparations can be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

In some embodiments, ubenimex is formulated for immediate release. Asused herein, “immediate release” means a drug formulation that providesfor the release of at least a majority of the drug within a relativelyshort period of time after administration (e.g., within about one hour).In some embodiments, an immediate release formulation provides for therelease of at least about 80% of the drug within about 30-60 minutesafter administration.

In some embodiments, ubenimex is formulated for sustained release orextended release. As used herein, “sustained release” and “extendedrelease” means a drug formulation that provides for gradual release of adrug over an extended period of time, and typically, although notnecessarily, results in substantially constant blood levels of a drugover an extended time period. In some embodiments, a sustained releaseformulation provides for a substantially constant blood level of theagent over a time period in the range of about 4 to about 12 hours,typically in the range of about 6 to about 10 hours. For example, asustained release formulation can provide a very gradual increase inblood level of a drug following administration such that peak bloodlevel is not reached until at least 4-6 hours have elapsed, with therate of increase of blood level drug approximately linear, followed by asustained period of peak blood levels and then by an equally gradualdecrease in blood levels at the end of the sustained release period.

In some embodiments, ubenimex is formulated for delayed release. As usedhere, “delayed release” refers to a drug formulation that, followingadministration to a patient, provides a measurable time delay beforedrug is released from the formulation into the patient's body.

In some embodiments, oral ubenimex formulations are formulated asimmediate release preparations, and are conveniently packaged, forexample, in unit dosage forms in the form of a pill, capsule, or tablet,which in turn may be in a pill bottle or blister packaging, or, forliquid formulations of the drug, in liquid compatible containers.Dosages and desired drug concentration of pharmaceutical compositions ofthe disclosure may vary depending on the particular use envisioned. Thedetermination of the appropriate dosage or route of administration iswell within the skill of one in the art. Suitable dosages are alsodescribed in Section III above. In some embodiments, ubenimex isprovided in oral form in a fixed dosage amount in a unit dosage formthat is a pill, tablet, or capsule containing amounts of ubenimexranging from 5-1000 mg, 10-800 mg, 20-750 mg, 30-600 mg, 40-500 mg,50-450 mg, 60-375 mg, 75-300 mg, 90-250 mg, or 100-150 mg. Theparticular unit dosage form will depend on the dose to be administered,which may depend on whether the patient is an adult or child or upon theseverity of the disease.

In some embodiments, most adult patients (60-100 kg or more) willreceive therapeutic benefit from a single dose in the range of 5-1000 mgper day, with some achieving full therapeutic effect with 5-450 mg atleast once daily, 10-225 mg twice daily, or 5-150 mg three times daily.However, minimal doses of 10 mg, 30 mg, and 150 mg administered on theseschedules (QD, BID, and TID) may be approved for clinical use. Somepatients will administer the prescribed dose with each meal. Somepatients will administer this dose before meals. Some patients willadminister this dose as a chronic medication; it is anticipated thatsome patients will take the drug every day for periods of 6 months orlonger.

The present disclosure also provides a variety of specific unit dosageforms suitable for use in the treatment methods described herein. Forexample, ubenimex can be administered in unit dosage forms containingfrom 5, 25, 30, 60, 90, or 150 mg of ubenimex, which are suitable fordelivery one, twice, or three times a day to provide the daily doseprescribed by the physician in accordance with this disclosure.Moreover, previous formulations and unit dosage forms of ubenimex can beused in the methods of the disclosure. Ubenimex has been marketed forthe treatment of cancer as an adjunct to chemotherapy agents to extentsurvival and maintain remission after treatment for acutenon-lymphocytic leukemia under the brand name Bestatin™ in Japan and isbeing studied in the clinic for treatment of PAH (see, WO 2013/142369and U.S. Pat. No. 9,233,089, each of which is incorporated herein byreference) and lymphedema (see, WO 2016/149126, incorporated herein byreference). In various embodiments of the disclosure, ubenimex,including but not limited to ubenimex in the Bestatin™ 10 mg or 30 mgunit dosage forms commercially available or in the forms being used inthe PAH and lymphedema clinical trials, is administered to a patientwith NASH.

In some embodiments, pharmaceutical packages and kits comprisingubenimex and a second therapeutic agent are provided. In someembodiments, the pharmaceutical package comprises ubenimex (e.g., in animmediate release, delayed release, or sustained release form) and asecond therapeutic agent, wherein the second therapeutic agent is afarnesoid X receptor (FXR) agonist (including but not limiting toobeticholic acid); a peroxisome proliferator-activator receptor (PPAR)agonist, e.g., a PPAR-alpha agonist, a PPAR-beta/delta agonist, or aPPAR-gamma agonist (including but not limited to elafibranor); aramchol;a caspase inhibitor (including but not limited to emricasan); a galectin3 inhibitor (including but not limited to GR-MD-02, GalectinTherapeutics); a MAPK5 inhibitor (including but not limited to GS-4997,Gilead Sciences); an FGF21 agonist (including but not limited toBMS-986036, Bristol-Myers Squibb); a LTD4 receptor antagonist (includingbut not limited to tipelukast); a niacin analog (including but notlimited to ARI 3037MO, Arisaph Pharmaceuticals); an ASBT inhibitor(including but not limited to volixibat); an apoptosis signal regulatingkinase 1 (ASK1) inhibitor; an angiotensin converting enzyme (ACE)inhibitor; an angiotensin receptor blocker (ARB, including but notlimited to telmisartan); a chemokine receptor inhibitor (e.g., CCR2and/or CCR5 inhibitors, including but not limited to cenicriviroc); athiozolidinedione (including but not limited to rosiglitazone andpioglitazone); a GLP-1 analog (including but not limiting tosemaglutide, liraglutide); a biguanide (e.g., metformin); or an NSAID.In some embodiments, the second therapeutic agent is not a chemotherapyagent.

In some embodiments, the pharmaceutical package or kit comprises unitdosage forms of ubenimex and further comprising unit dosage forms of asecond therapeutic agent, wherein the second therapeutic agent is a FXRagonist, a PPAR agonist, aramchol, a caspase inhibitor, a galectin 3inhibitor, a MAPK5 inhibitor, a FGF21 agonist, a LTD4 receptorantagonist, a niacin analog, an ASBT inhibitor, an ASK1 inhibitor, anACE inhibitor, an angiotensin receptor blocker, a chemokine receptorinhibitor, a thiozolidinedione, a GLP-1 analog, a biguanide, or anNSAID.

In some embodiments, the pharmaceutical package or kit is for use intreating NASH. In some embodiments, the pharmaceutical package or kit isfor use in delaying or preventing the progression of NAFLD to NASH in asubject having NAFLD. In some embodiments, the pharmaceutical package orkit further comprises instructional materials for use according to amethod disclosed herein. While the instructional materials typicallycomprise written or printed materials they are not limited to such. Anymedium capable of storing such instructions and communicating them to anend user is contemplated by this invention. Such media include, but arenot limited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD-ROM), and the like. Suchmedia may include addresses to Internet sites that provide suchinstructional materials.

V. Combination Therapies for the Treatment of NASH

In another aspect, ubenimex is administered in combination with one ormore additional therapeutic agents. In some embodiments, a method oftreating NASH (and/or NAFLD) may further include in accordance with thedisclosure a combination therapy in which a patient in need of treatmentis administered ubenimex in combination with one or more drugs approvedfor the treatment of NASH (although there are none today, but such drugsincludes drugs in clinical testing today that are later approved, ifany), and/or an associated condition of NASH, or a combination thereof.Combination therapies for the treatment of NASH may further includeadministration of ubenimex in combination with one or more lifestylechanges, such as exercise to reduce body weight and/or activities toimprove physical and/or mental health.

In some embodiments, ubenimex is administered in combination with one ormore of a farnesoid X receptor (FXR) agonist (including but not limitingto obeticholic acid; EDP-305, Enanta Pharmaceuticals; and GS-9674,Gilead Sciences); a peroxisome proliferator-activator receptor (PPAR)agonist, e.g., a PPAR-alpha agonist, a PPAR-beta/delta agonist, and/or aPPAR-gamma agonist (including but not limited to elafibranor); aramchol;a caspase inhibitor (including but not limited to emricasan); a galectin3 inhibitor (including but not limited to GR-MD-02, GalectinTherapeutics); a MAPK5 inhibitor (including but not limited to GS-4997,Gilead Sciences); an FGF19 agonist (including but not limited to NGM282,NGM Bio); an FGF21 agonist (including but not limited to BMS-986036,Bristol-Myers Squibb); a LTD4 receptor antagonist (including but notlimited to tipelukast); a niacin analog (including but not limited toARI 3037MO, Arisaph Pharmaceuticals); an ASBT inhibitor (including butnot limited to volixibat); an apoptosis signal regulating kinase 1(ASK1) inhibitor; a chemokine receptor inhibitor (e.g. CCR2 and/or CCR5inhibitors, including but not limited to cenicriviroc); athiozolidinedione (including but not limited to rosiglitazone andpioglitazone); a GLP-1 analog (including but not limiting tosemaglutide, liraglutide); a biguanide (e.g. metformin); a nonsteroidalanti-inflammatory drug (“NSAID”) (e.g., aceclofenac, acemetacin,aspirin, celecoxib, dexibuprofen, dexketoprofen, diclofenac, etodolac,etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indometacin,ketoprofen, mefenamic acid, meloxicam, nabumetone, naproxen, sulindac,tenoxicam, and tiaprofenic acid); an antidiabetic (e.g., biguanides,sulfonylureas, meglitinides, thiazolidinediones, tiazolidinediones,pioglitazone, dipeptidyl peptidase IV inhibitors, and a-glucosidaseinhibitors); a lipid-lowering agent (e.g., atorvastatin, rosuvastatin,fluvastatin, lovastatin, simvastatin, pravastatin, pitavastatin, niacin,fenofibrate, fenofibric acid, gemfibrozil, and omega-3 polyunsaturatedfatty acid), an angiotensin converting enzyme (ACE) inhibitor (e.g.,captopril, enalapril, lisinopril, benazepril, fosinopril, quinapril, andramipril); an angiotensin II receptor blocker (“ARBs”) (e.g.,irbesartan, losartan, olmesartan, valsartan, and telmisartan),anti-angiogenic agents (e.g., sorafenib); vitamin E; an antiplateletagent (e.g., aspirin); an anti-obesity or appetite suppressant (e.g.,phentermine, orlistat, lorcaserin, phendimetrazine, methamphetamine,benzphatamine, diethylpropion, sibutramine, topiramate, and bupropion);an psychosomatic agent (e.g., antidepressants, and antipsychotics), aAcetyl-CoA carboxylase inhibitor (including but not limited to GS-0976,Gilead); a PDE-5 inhibitor; or a deacetylase sirtuin stimulator. In someembodiments, ubenimex is not administered in combination with achemotherapy agent.

In one embodiment, the present disclosure comprises a combinationtherapy wherein ubenimex is administered in combination with an ACEinhibitor. In one embodiment, ubenimex is administered in combinationwith telmisartan. In one embodiment, ubenimex is administered incombination with one or more CCR2/CCR5 inhibitors. In one embodiment,ubenimex is administered in combination with cenicriviroc. In oneembodiment, ubenimex is administered in combination with athiazolidinedione, for example, rosiglitazone or pioglitazone. In oneembodiment, ubenimex is administered in combination with metformin. Inone embodiment, ubenimex is administered in combination with an FXRagonist, including but not limited to Ocaliva (obeticholic acid), whichis approved for biliary cholangitis. In one embodiment, ubenimex isadministered in combination with a PPAR alpha/delta agonist, includingbut not limited to Elafibranor, currently in clinical trials. In someembodiments, ubenimex is administered in combination with a compounddisclosed in Musso et al., Nature Reviews Drug Discovery, 2016,15:249-274.

In some embodiments, ubenimex is administered in combination withtelmisartan or losartan. In some embodiments, ubenimex is administered adosage of about 10 mg to about 450 mg and telmisartan is administered ata dosage of about 20 to 80 mg (e.g., QD or BID).

In some embodiments, ubenimex is administered in combination withobeticholic acid (OCA). In some embodiments, ubenimex is administered adosage of about 10 mg to about 450 mg and OCA is administered at adosage of 10, 25, or 40 mg (e.g., QD or BID).

In some embodiments, ubenimex is administered in combination withvolixibat. In some embodiments, ubenimex is administered a dosage ofabout 10 mg to about 450 mg and volixibat is administered at a dosage ofabout 5 to 20 mg (e.g., QD or BID).

In some embodiments, ubenimex is administered in combination witharamchol. In some embodiments, ubenimex is administered a dosage ofabout 10 mg to about 450 mg and aramchol is administered at a dosage ofabout 100 to 300 mg (e.g., QD or BID).

In some embodiments, ubenimex is administered in combination withelafibranor.

In some embodiments, ubenimex is administered a dosage of about 10 mg toabout 450 mg and elafibranor is administered at a dosage of about 120 mg(e.g., QD or BID).

The beneficial effect of the combination or ubenimex and a secondtherapeutic agent may include, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Such combination therapies can result in an improved parametersuch as, but not limited to, improved NASH scores (e.g., as measured bythe NAFLD Activity Score), decreased fibrosis scores, and decreasedserum alanine aminotransferase (ALT) levels; improved insulinsensitivity in adipose tissue (including but not limited to byactivating PPAR gamma); biochemical and histological improvements inNASH; improved (decreased) insulin resistance; and improvements insteatosis, inflammation, and fibrosis.

Administration of therapeutic agents in combination typically is carriedout over a defined time period (e.g., over a period of days, weeks, ormonths depending upon the combination selected). Combination therapyincludes administration of at least two different therapeutic agents ina sequential manner, wherein each therapeutic agent is administered at adifferent time, as well as administration of at least two differenttherapeutic agents in a substantially simultaneous manner. Substantiallysimultaneous administration can be accomplished, for example, byadministering to the subject a single capsule having a fixed ratio ofeach therapeutic agent or in separate capsules for each of thetherapeutic agents. In some embodiments, the ubenimex and the secondtherapeutic agent are formulated separately. In some embodiments, theubenimex and the second therapeutic agent are formulated in a singlecomposition.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route, including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. The twodifferent therapeutic agents can be administered by the same route or bydifferent routes. For example, a first therapeutic agent of thecombination selected may be administered by intravenous injection whilethe second therapeutic agent of the combination may be administeredorally. Alternatively, for example, all therapeutic agents may beadministered orally or all therapeutic agents may be administered byintravenous injection.

For the combination therapies disclosed herein, it is contemplated thateach agent can be administered in an “immediate release” manner or in a“controlled release” or “delayed release” manner. When the additionalactive agent is an anti-inflammatory, for instance, any dosage formcontaining both active agents, i.e., both the ubenimex and theanti-inflammatory, can provide for immediate release or controlledrelease of the anti-inflammatory, and either immediate release orcontrolled release of the ubenimex.

As a non-limiting example, a combination dosage form for once-dailyadministration may contain in the range of about 5 mg to about 450 mg ofubenimex in a controlled release (e.g., sustained or extended release)or immediate release form, and a secondary agent in immediate releaseform, or in controlled release form, with the additional active agentpresent in an amount that provides a weight ratio of the ubenimex to thesecondary agent. In other formulations, two or more secondary agents,which may or may not be in the same class of drug (e.g.,anti-inflammatories), can be present in combination, along withubenimex. In such a case, the effective amount of either or eachindividual secondary agents present will generally be reduced relativeto the amount that would be required if only a single added agent wereused.

Combination therapy also includes the administration of the differenttherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orphysical therapy). Where a combination therapy comprises a non-drugtreatment, the non-drug treatment may be conducted at any suitable timeso long as a beneficial effect from the co-action of the combination ofthe therapeutic agents and non-drug treatment is achieved. For example,in appropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1: Anti-NASH Efficacy of Ubenimex in an Animal Model

The STAM™ mouse is a model for NASH created by a combination of chemicaland dietary interventions in C57BL/6 mice that manifest NASH at about 8weeks, which progresses to fibrosis at about 12 weeks and thenprogresses to HCC at about 16 weeks. See, Saito et al., Sci. Rep, 2015,5:12466. This model was used in the study described herein todemonstrate the efficacy of ubenimex in treating NASH.

In the study, the control groups were as follows: (i) Untreated DiseaseControl—a group of STAM mice injected with streptozotocin (STZ,Sigma-Aldrich, USA) at 2 days old, and fed a high fat diet (HFD, 57 kcal% fat, Cat # HFD32, CLEA Japan, Japan) starting at 4 weeks old; and (ii)Positive Control—a group of SIAM mice treated with telmisartan (10 mg/kgQD from 7 to 9 weeks). In addition to these control groups, test groupsof STAM mice treated with a low dose of ubenimex (5 mg/kg ubenimex BIDfrom 7 to 9 weeks) or a high dose of ubenimex (25 mg/kg BID from 7 to 9weeks), were included in the study. The low dose is equivalent to ahuman dose of 30 mg BID. The high dose is equivalent to a human dose of150 mg BID. The viability, clinical signs and behavior were monitoreddaily, including body weight, signs of toxicity, moribundity andmortality. The mice were maintained in a SPF facility under controlledconditions of temperature (23±2° C.), humidity (45±10%), lighting(12-hour artificial light and dark cycles; light from 8:00 to 20:00) andair exchange. A high pressure was maintained in the experimental room toprevent contamination of the facility. The mice were housed in TPX cages(CLEA Japan) with a maximum of 4 mice per case.

All groups of mice were sacrificed at 9 weeks. Once sacrificed, the bodyweight of each mouse was recorded. The liver of each mouse was removedand weighed. The liver-to-body weight ratio was determined for eachmouse. Blood serum levels of CK-18, ALT, and liver triglycerides weredetermined for each mouse. The livers were then sectioned and stainedwith hematoxylin and eosin (“HE”) stain, as well as Sirius red staining,to determine liver steatosis (ballooning) and fibrosis, respectively.

Table 1 below shows the results of the body and liver weight testing,which generally indicated that the ubenimex treatment was safe.Generally, the telmisartan group showed a significant decrease in meanbody weight on the day of sacrifice compared with the control group. Thelow and high dose groups tended to decrease in mean body weight on theday of sacrifice, as compared with the control group. The high dose, lowdose, and telmisartan groups showed significant decreases in mean liverweight compared with the control group. The high dose and telmisartangroups tended to decrease mean liver-to-body weight ratio as comparedwith the control group. There was no significant difference in meanliver-to-body weight ratio between the control group and the low dosegroup.

TABLE 1 Body weight and liver weight Disease- Ubenimex, Ubenimex,Telmisartan, Parameter control 5 mg/kg BID 25 mg/kg BID 10 mg/kg QD(mean ± SD) (n = 8) (n = 6) (n = 7) (n = 8) Body weight (g) 18.8 ± 1.616.6 ± 2.8 17.7 ± 1.1 15.8 ± 2.0 Liver weight (mg) 1420 ± 165 1206 ± 1431190 ± 83  1027 ± 76  Liver-to-body weight  7.6 ± 1.1  7.4 ± 1.2  6.7 ±0.3  6.6 ± 0.6 ratio (%)

Table 2 below shows that the high and low dose ubenimex groups and thepositive control telmisartan group all showed significant reductions inNAFLD activity score (NAS) as compared with the control group.

TABLE 2 NAFLD Activity Score Score Lobular Hepatocyte Steatosisinflammation ballooning NAS Group n 0 1 2 3 0 1 2 3 0 1 2 (mean ± SD)Disease-control 8 — 7 1 — — — 6 2 — 2 6 5.1 ± 1.0 Ubenimex, 6 1 5 — — —1 3 2 4 2 — 3.3 ± 0.5 5 mg/kg BID Ubenimex, 7 — 7 — — — — 5 2 5 2 — 3.6± 0.5 25 mg/kg BID Telmisartan, 8 3 5 — — — 2 4 2 8 — — 2.6 + 0.9 10mg/kg QD Definitions of NAS Components Item Score Extent Steatosis 0    <5% 1  5-33% 2 >33-66% 3    >66% Lobular 0 No foci inflammation 1 <2foci/200x 2 2-4 foci/200x  3 >4 foci/200x Hepatocyte 0 None ballooning 1Few balloon cells 2 Many cells/prominent ballooning

As shown in Table 2, ubenimex therapy demonstrated significant reductionin NAS as compared to the negative control group. NAS is one of theclinical endpoints for assessing the activity of NASH (Sanyal et al.,Hepatology, 2011, 54:344), and thus is the key preclinical endpoint inclinical translation. The improvement of NAS by ubenimex was, at thistime point, attributable to the changes in hepatocyte ballooning, whichwas significantly decreased compared with the control group. Rangwalareported the close association of hepatocyte ballooning and NASH-relatedfibrosis (Rangwala et al., J. Pathol, 2011, 224:401), which indicatesthat continued treatment should have a positive impact on fibrosis.Consistent with this, treatment with ubenimex was observed in the lowdose (e.g., 5 mg/kg BID) group to tend to reduce the pathologicaldeposition of collagen in the liver, as demonstrated by Sirius redstaining. Thus, reduction of hepatocyte ballooning in the high and lowdose ubenimex groups is indicative not only of a positive treatmentimpact but strong support for continuing therapy to increase theanti-fibrosis effects of this treatment.

As shown in Table 3 below, no significant effect on plasma ALT or livertriglyceride levels were seen in the test groups, although thetelmisartan positive control group reflected a trend of decreased levelsof both relative to the disease (negative) control group. It is believedthat continued administration of ubenimex would reduce these levels.

TABLE 3 Biochemistry Disease- Ubenimex, Ubenimex, Telmisartan, Parametercontrol 5 mg/kg BID 25 mg/kg BID 10 mg/kg QD (mean ± SD) (n = 8) (n = 6)(n = 7) (n = 8) Plasma ALT (U/L) 59 ± 27 59 ± 22 62 ± 11 44 ± 13 Livertriglyercide 46.4 ± 16.5 50.0 ± 22.7 34.8 ± 22.2 22.4 ± 7.5  (mg/gliver)

As shown in Table 4, liver sections from the disease control groupshowed increased collagen deposition in the pericentral region of liverlobule. The telmisartan group showed a significant decrease in thefibrosis area (Sirius red-positive area) as compared with the controlgroup. The low dose group showed a tendency to decrease the fibrosisarea compared to the disease control group, but this was not seen in thehigh dose group (no significant difference in fibrosis area between thecontrol group and the high dose group). In this proof-of-concept studydesigned to evaluate efficacy of ubenimex on NASH only, the animals weresacrificed at age of 9 weeks while full development of liver fibrosis inthis animal model typically occurs at age of 12 weeks, it is believedthat longer duration of treatment in animals allowed for fulldevelopment of fibrosis would have resulted in a more profoundtherapeutic effect in both treatment dose groups.

TABLE 4 Fibrosis Area Disease- Ubenimex, Ubenimex, Telmisartan,Parameter control 5 mg/kg BID 25 mg/kg BID 10 mg/kg QD (mean ± SD) (n =8) (n = 6) (n = 7) (n = 8) Sirius red-positive area 1.02 ± 0.32 0.80 ±0.16 1.05 ± 0.14 0.71 ± 0.15 (%)

Plasma CK-18 (in units of mlU/mL) was measured and found to besignificantly lower in the low and high dose ubenimex groups (238.5±7.7and 230.8±28.6, respectively) and relatively unchanged between thecontrol and telmisartan groups (292.3±28.5 and 284.5±38.4,respectively). These results convincingly demonstrate that ubenimex hasa different and potentially more potent therapeutic benefit thantelmisartan.

Plasma LTB4 levels were measured but were not considered meaningful inthat while the ubenimex low dose and telmisartan groups showed sometendency to increase plasma LTB4 relative to the control, the controland ubenimex high dose levels measured were not statistically different,and there was significant variability in all but the control group.

These results support continued studies of ubenimex in NASH animalmodels and human clinical trials.

Example 2: Ubenimex Human Clinical Trials

Two or more groups of human subjects affected by NASH are eachadministered a pharmaceutically effective dose of ubenimex comprising 30mg, with one group receiving QD administration (i.e., a total daily doseof 30 mg) and the other BID (i.e., a total daily dose of 60 mg). A humancontrol group affected by NASH is administered a placebo. The study isconducted for approximately 6 months, during which the participants aremonitored for improvements in at least one of NAFLD Activity Score,Steatosis Score, Inflammation Score, and Ballooning Score. Treatment isanticipated to improve one or more of said scores.

Example 3: In Viva Efficacy Study of Ubenimex in STAM Model of NASH withFibrosis

This study examined the effects of ubenimex in a STAM model of NASHfibrosis at multiple dosing regimens.

Materials and Methods

NASH was induced in 40 male mice by a single subcutaneous injection of200 μg streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days afterbirth and feeding with a high fat diet (HFD, 57 kcal % fat, Cat # HFD32,CLEA Japan Inc., Japan) after 4 weeks of age.

Ubenimex or telmisartan was administered orally in a volume at 10 mL/kg.Ubenimex was administered at a dose of 5 mg/kg once daily or twicedaily. Telmisartan was administered at a dose of 5 mg/kg once daily.

C57Bl/6 mice (14-day-pregnant females) were obtained from Japan SLC,Inc. (Japan). All animals used in the study were housed and cared for inaccordance with the Japanese Pharmacological Society Guidelines forAnimal Use. The animals were maintained in a SPF facility undercontrolled conditions of temperature (23±2° C.), humidity (45±10%),lighting (12-hour artificial light and dark cycles; light from 8:00 to20:00) and air exchange. A high pressure was maintained in theexperimental room to prevent contamination of the facility. The micewere housed in TPX cages (CLEA Japan) with a maximum of 4 mice per case.Sterilized solid HFD and pure water were provided ad libitum.

Measurement of plasma biochemistry: For plasma biochemistry, blood wascollected in polypropylene tubes with anticoagulant (Novo-Heparin,Mochida Pharmaceutical Co. Ltd., Japan) and centrifuged at 1,000×g for15 minutes at 4° C. The supernatant was collected and stored at −80° C.until use. Plasma ALT level was measured by FUJI DRI-CHEM 7000(Fujifilm, Japan).

Measurement of liver triglyceride content: Liver total lipid-extractswere obtained by Folch's method (Folch et al., J. Biol Chem, 1957,226:497). Liver samples were homogenized in chloroform-methanol (2:1v/v) and incubated overnight at room temperature. After washing withchloroform-methanol-water (8:4:3 v/v/v), the extracts were evaporated todryness and dissolved in isopropanol. Liver triglyceride content wasmeasured by Triglyceride E-test (Wako Pure Chemical Industries, Ltd.,Japan).

Histological analyses: For HE staining, sections were cut from paraffinblocks of liver tissue prefixed in Bouin's solution and stained withLillie-Mayer's hematoxylin (Moto Pure Chemicals Co., Ltd., Japan) andeosin solution (Wako Pure Chemical Industries, Ltd., Japan). NAFLDActivity Score (NAS) was calculated according to the criteria of Kleiner(Kleiner et al., Hepatology, 2005, 41:1313). To visualize collagendeposition, Bouin's fixed liver sections were stained using picro-Siriusred solution (Waldeck, Germany). For quantitative analysis of fibrosisarea, bright field images of Sirius red-staining sections were capturedaround the central vein using a digital camera (DFC295; Leica, Germany)at 200-fold magnification, and the positive areas in 5 fields/sectionwere measured using ImageJ software (National Institutes of Health,USA).

Statistical tests: Statistical analyses were performed using BonferroniMultiple Comparison Test on GraphPad Prism 6 (GraphPad Software Inc.,USA). P values<0.05 were considered statistically significant. A trendor tendency was assumed when a one-tailed t-test returned P values<0.1.Results were expressed as mean±SD.

Experimental Design and Treatment

Study groups: The following study groups were used. Group 1 (Vehicle): 8NASH mice were orally administered vehicle (RO water) in a volume of 10mL/kg twice daily from 6 to 11 weeks of age. Group 2 (Ubenimex QD for 5weeks): 8 NASH mice were orally administered vehicle supplemented withubenimex at a dose of 5 mg/kg once daily from 6 to 11 weeks of age.Group 3 (Ubenimex BID for 5 weeks): 8 NASH mice were orally administeredvehicle supplemented with ubenimex at a dose of 5 mg/kg twice daily from6 to 11 weeks of age. Group 4 (Ubenimex BID for 2 weeks): 8 NASH micewere orally administered vehicle supplemented with ubenimex at a dose of5 mg/kg twice daily from 9 to 11 weeks of age. Group 5 (Telmisartan for5 weeks): 8 NASH mice were orally administered pure water supplementedwith telmisartan at a dose of 5 mg/kg once daily from 6 to 11 weeks ofage.

Animal monitoring and sacrifice: The viability, clinical signs, andbehavior were monitored daily. Body weight was recorded before thetreatment. Mice were observed for significant clinical signs oftoxicity, moribundity and mortality approximately 60 minutes after eachadministration. The animals were sacrificed by exsanguination throughdirect cardiac puncture under isoflurane anesthesia.

Results

Mean body weight of the day of sacrifice, mean liver weight, and meanliver-to-body weight ratio are shown in Table 5 below for all fivegroups. The mean body weight on the day of sacrifice was significantlydecreased in the ubenimex BID for 5 weeks group and telmisartan positivecontrol group as compared with the vehicle group. The mean body weighton the day of sacrifice tended to decrease in the ubenimex QD group ascompared with the vehicle group. There were no significant differencesin mean body weight on the day of sacrifice between the vehicle groupand the ubenimex BID for 2 weeks group. For liver weight, thetelmisartan group showed a significant decrease in mean liver weight ascompared with the vehicle group. Mean liver weight tended to decrease inthe ubenimex QD and ubenimex BID for 5 weeks groups as compared with thevehicle group. There were no significant differences in mean liverweight between the vehicle group and the ubenimex BID for 2 weeks group.The telmisartan group showed a significant decrease in meanliver-to-body weight ratio as compared with the vehicle group. Therewere no significant differences in mean liver-to-body weight ratiobetween the vehicle group and the ubenimex treatment groups.

TABLE 5 Body weight and liver weight Group 2 Group 3 Group 4 Group 5Group 1 (UBX QD; (UBX BID; (UBX BID; (Telmisartan; Parameter (vehicle) 5weeks) 5 weeks) 2 weeks) 5 weeks) (mean ± SD) (n = 6) (n = 6) (n = 6) (n= 6) (n = 6) Body weight (g) 22.3 ± 3.1 19.9 ± 1.5 18.7 ± 1.6 21.3 ± 2.218.3 ± 1.9 Liver weight 1806 ± 263 1526 ± 191 1539 ± 258 1603 ± 321 1105± 105 (mg) Liver-to-body  8.1 ± 1.1  7.7 ± 0.9  8.3 ± 1.2  7.5 ± 0.9 6.1 ± 0.5 weight ratio (%)

As shown in Table 6 below, plasma ALT level tended to decrease in thetelmisartan group as compared to the vehicle group, although thisdifference was not statistically significant. There were no significantdifferences in plasma ALT levels between the vehicle group and theubenimex treatment groups.

Liver triglyceride content tended to decrease in the telmisartan groupas compared to the vehicle group (Table 6), although this difference wasnot statistically significant. There were no significant differences inliver triglyceride content between the vehicle group and the ubenimextreatment groups.

TABLE 6 Biochemistry Group 2 Group 3 Group 4 Group 5 Group 1 (UBX QD;(UBX BID; (UBX BID; (Telmisartan; Parameter (vehicle) 5 weeks) 5 weeks)2 weeks) 5 weeks) (mean ± SD) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6)Plasma ALT 56 ± 33 85 ± 64 52 ± 21 47 ± 24 36 ± 11 (U/L) Liver 60.4 ±22.6 58.6 ± 24.4 59.6 ± 21.4 62.0 ± 33.5 24.6 ± 8.4  triglyceride (mg/gliver)

FIG. 1A and Table 7 show that NAFLD activity score (NAS) tended todecrease in the ubenimex treatment groups and positive controltelmisartan group as compared with the control group. The NAFLD activityscore is determined from a combination of steatosis score, inflammationscore, and hepatocyte ballooning score. As shown in FIGS. 1B-D and Table7, although no significant differences were observed between the vehiclegroup and the ubenimex therapy or telmisartan groups with respect to thesteatosis score or inflammation score, hepatocyte ballooning tended todecrease in the ubenimex treatment groups and positive controltelmisartan group as compared to the vehicle group. Using the studentt-test, a significant decrease was observed for hepatocyte ballooningscore between the group treated with ubenimex BID for 5 weeks and thecontrol group (p=0.0493). As discussed in Example 1 above, Rangwalareported the close association of hepatocyte ballooning and NASH-relatedfibrosis, which indicates that continued treatment should have apositive impact on fibrosis. Consistent with this hypothesis, treatmentwith QD ubenimex for 5 weeks was observed to tend to decrease thefibrosis area, as demonstrated by Sirius red staining (see Table 8below). The reduction of hepatocyte ballooning in the ubenimex treatmentgroups is indicative of a positive therapeutic effect by ubenimex in thetreatment of NASH.

TABLE 7 NAFLD Activity Score Score Lobular Hepatocyte Steatosisinflammation ballooning NAS Group n 0 1 2 3 0 1 2 3 0 1 2 (mean ± SD)Vehicle 6 — 5 1 — — — 4 2 — 5 1 4.7 ± 0.8 UBX QD; 5 weeks 6 — 6 — — — 13 2 2 4 — 3.8 ± 0.8 UBX BID; 5 weeks 6 1 4 1 — — — 2 4 5 — 1 4.0 ± 1.7UBX BID; 2 weeks 6 1 4 1 — — 4 2 4 1 1 3.8 ± 1.0 Telmisartan; 5 weeks 61 5 — — 1 — 3 2 3 3 — 3.3 ± 1.0 Definitions of NAS components are shownin Table 2 above.

Liver sections from the vehicle control group showed increased collagendeposition in the pericentral region of liver lobule. As shown in Table8 below, the telmisartan positive group showed a significant decrease inthe fibrosis area (Sirius red-positive area) as compared with thevehicle group. The fibrosis area tended to decrease in the ubenimex QDgroup as compared with the vehicle group.

TABLE 8 Fibrosis area. Group 2 Group 3 Group 4 Group 5 Group 1 (UBX QD;(UBX BID; (UBX BID; (Telmisartan; Parameter (vehicle) 5 weeks) 5 weeks)2 weeks) 5 weeks) (mean ± SD) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6)Sirius red- 1.03 ± 0.45 0.75 ± 0.16 0.88 ± 0.22 0.87 ± 0.14 0.57 ± 0.14positive area (%)

In summary, treatment with ubenimex once daily for a longer period oftime (5 weeks) showed decreasing trends in NAS scores and fibrosis, asshown by the data in FIG. 1A and in Tables 7 and 8. Treatment withubenimex twice daily for a shorter period of time (2 weeks) showeddecreasing trends in NAS scores, as shown by the data in FIG. 1A and inTable 7. As shown in FIG. 1D and Table 7, the improvement in NAS scoreswas characterized by a reduction in the hepatocyte ballooning score.

While this disclosure has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the disclosureencompassed by the appended claims.

All publications, patents, patent applications, or other documents citedherein are hereby incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, or other document was individually indicated to beincorporated by reference for all purposes.

What is claimed is:
 1. A method of treating non-alcoholicsteatohepatitis (NASH), the method comprising administering to a subjectin need of treatment a therapeutically effective amount of ubenimex. 2.The method of claim 1, wherein the subject has early-stage ormiddle-stage NASH.
 3. The method of claim 1, wherein the ubenimex isadministered at a total daily dose in the range of 5 mg to 1000 mg. 4.The method of claim 1, wherein the ubenimex is administered at a dailydose of about 5 mg to about 450 mg.
 5. The method of claim 1, whereinthe ubenimex is administered for at least 4 weeks.
 6. The method ofclaim 5, wherein the ubenimex is administered for at least 12 weeks. 7.The method of claim 1, wherein treatment results in a reduction inplasma CK-18 levels in the subject.
 8. The method of claim 1, whereintreatment results in a reduction in hepatocyte ballooning in thesubject.
 9. The method of claim 1, wherein the ubenimex is administeredin combination with a second therapeutic agent.
 10. The method of claim9, wherein the second therapeutic agent is a farnesoid X receptor (FXR)agonist, a peroxisome proliferator-activator receptor (PPAR) agonist,aramchol, a caspase inhibitor, a galectin 3 inhibitor, amitogen-activated protein kinase 5 (MAPK5) inhibitor, a fibroblastgrowth factor 19 (FGF19) agonist, a FGF21 agonist, a leukotriene D4(LTD4) receptor antagonist, a niacin analog, an apical sodium bile acidcotransporter (ASBT) inhibitor, an apoptosis signal regulating kinase 1(ASK1) inhibitor, an angiotensin converting enzyme (ACE) inhibitor, anangiotensin receptor blocker, a chemokine receptor inhibitor, athiozolidinedione, a GLP-1 analog, a biguanide, or a non-steroidalanti-inflammatory drug (NSAID).
 11. A method of delaying or preventingthe progression of non-alcoholic fatty liver disease (NAFLD) to NASH ina subject having NAFLD, the method comprising administering to thesubject a therapeutically effective amount of ubenimex.
 12. The methodof claim 11, wherein treatment results in a reduction in hepatocyteballooning in the subject.
 13. The method of claim 11, wherein theubenimex is administered at a daily dose in the range of 5 mg to 1000mg.
 14. The method of claim 11, wherein the ubenimex is administered ata daily dose of about 5 mg to about 450 mg.
 15. The method of claim 11,wherein the ubenimex is administered for at least 4 weeks.
 16. Themethod of claim 15, wherein the ubenimex is administered for at least 12weeks.
 17. A method of decreasing inflammation and/or fibrosis in asubject having NASH, the method comprising administering to the subjecta therapeutically effective amount of ubenimex for at least 24 weeks.18. The method of claim 17, wherein the ubenimex is administered for atleast 48 weeks.
 19. The method of claim 11, wherein the ubenimex isadministered in combination with a second therapeutic agent.
 20. Themethod of claim 19, wherein the second therapeutic agent is a FXRagonist, a PPAR agonist, aramchol, a caspase inhibitor, a galectin 3inhibitor, a MAPK5 inhibitor, a FGF19 agonist, a FGF21 agonist, a LTD4receptor antagonist, a niacin analog, an ASBT inhibitor, an ASK1inhibitor, an ACE inhibitor, an angiotensin receptor blocker, achemokine receptor inhibitor, a thiozolidinedione, a GLP-1 analog, abiguanide, or an NSAID.